Method

ABSTRACT

The present invention relates to a method for determining a mass changing event in a very small amount of a material of interest (eg a chemical or biological material of interest). The mass changing event may be for example specific binding or nucleotide complementation and may be differentiated over other (non-specific) events.

The present invention relates to a method for determining a masschanging event in a material of interest (eg a chemical or biologicalmaterial of interest).

Mass changes may occur when two or more molecules associate ordissociate in a specific interaction (eg in a protein binding system orDNA hybridisation). However associative or dissociative interactions ofa non-specific nature (eg resulting from weak chemical forces such asLondon forces and dispersion phenomena) also lead to a mass change andas a consequence manifest themselves as undesirable background noise.This complicates interpretation of the measurement and it may be verydifficult to determine the extent of the interaction between the twomolecules and their subsequent behaviour. The extent of the interactionis (for example) of particular importance in the analysis of DNAfragments where the differences in the nucleotide sequences arefrequently very small. Moreover, determining the molecular behaviour ofa molecule such as a protein is crucial to the overall understanding ofbiological processes.

In biological fluids, there may be numerous proteins whose nature andbehaviour are unknown. A number of such proteins may interactindiscriminately with a biosensor in events often described as“non-specific binding events”. These non-specific binding eventscontribute to a signal which cannot be differentiated from the signalwhich results from the desired interaction (“the specific bindingevent”). Such non-specific binding events hamper biotechnologists andlimit the utility of biochemical systems based on detector/sensorplatforms by reducing the degree of confidence in the measurement (andin some circumstances rendering any measurement impossible). As aresult, users are forced to carry out rigorous ‘clean up’ procedures onbiological fluids to remove unwanted proteins and other extraneousmaterial before seeking to measure the specific binding event(s).

Many inorganic and organic materials exhibit specific behaviour as aconsequence of exposure to physical stimuli (eg temperature, solvationor pH) or to chemical stimuli. These stimuli are frequently unrelated tothe stimulus of interest. Even when such stimuli are related to thestimulus of interest the measured response may be difficult (if notimpossible) to interpret. This leads at best to an inaccuracy in themeasurement and at worst to complete breakdown of the measurementmethod.

The present invention seeks to improve the determination of the masscharacteristics of a material of interest by exploiting the sensitivityof certain sensor devices to mass changing events at the molecularlevel. More particularly, the present invention relates to a method fordetermining a mass changing event in the material of interest bymeasuring the temporal response of a sensor device to which the materialof interest is exposed.

Thus viewed from one aspect the present invention provides a method fordetermining a mass changing event in a material of interest in alocalised environment, said method comprising:

-   -   (A) providing a sensor device having a sensor component capable        of exhibiting a measurable response to a change in the localised        environment caused by the mass changing event in the material of        interest therein;    -   (B) introducing the material of interest into the localised        environment;    -   (C) inducing the mass changing event in the material of        interest;    -   (D) generating an output from the sensor component over a        temporal range;    -   (E) measuring the response of a characteristic of the output        over the temporal range; and    -   (F) relating the response of the characteristic of the output        over the temporal range to the mass changing event.

The temporal response of the characteristic of the output of the sensorcomponent depends crucially upon the type of mass changing event. It isthe sensitivity of the sensor component leading to the measurablevariation in the temporal response which enables different mass changingevents to be differentiated.

The sensor component may be capable of exhibiting a measurable responsein a parameter selected from effective refractive index, a dielectricconstant, a viscoelastic property, a frequency of oscillation, a thermalabsorption/desorption parameter, the permeability, the absorption ofenergy or of energetic particles (such as x-rays, gamma rays, β-rays,electrons, neutrons, ions, light, microwaves, acoustic waves) or theparticle size. For example, the sensor device may be one or more of thefollowing types: surface plasmon resonance sensor devices, resonantmirror sensor devices, acoustic sensor devices (such as quartz crystaland surface acoustic wave devices (by using frequency decay techniquesfor example)) or electrical sensor devices (capable of measuringimpedance at (for example) RF or microwave frequencies). Preferably theparameter is the effective refractive index.

In a preferred embodiment, step (D) comprises:

-   (D) irradiating the sensor component with electromagnetic radiation    to generate an output over a temporal range.

The mass changing event may be a chemical (ie non-physical) masschanging event.

When molecules interact (eg bond) in a specific manner there is ingeneral an increase in molecular density whilst when molecules interactin a non-specific manner there is no significant change in moleculardensity. By measuring the temporal response, the present invention candistinguish such events. Thus in a preferred embodiment, the masschanging event is a specific molecular (or atomic) interaction. Forexample, the mass changing event is a specific associative ordissociative molecular interaction.

Without wishing to be bound by any theoretical considerations, it isnoted that (in general) when two or more molecules interact (eg bond) ina chemically active manner, molecular density changes (typicallyincreasing) with respect to the molecular density of the individualmolecules. The factors effecting the molecular density after the bondingevent (or binding event as it is often referred to) include the strengthand nature of the bond(s) and any consequential change in conformation.If (on the other hand) two molecules interact in a chemically inactivemanner, the combined molecular mass may increase but the moleculardensity stays largely unchanged because the overall volume alsoincreases. By measuring the temporal response, the present inventionmakes it possible to differentiate the two types of interaction.Similarly, the breaking of a chemical bond will result in a moleculardensity change (typically decreasing) which can be differentiated from achemically inactive change. By providing a unique signature of the typeof interaction taking place, the invention will be of significantutility, in particular in the field of biosensing where the backgroundmeasurements often severely limit the utility of the measurement.

In a preferred embodiment of the method of the invention, the masschanging event is a binding event (eg chemical, biochemical orbiological binding event). The mass changing event may be a specificbinding event or a non-specific binding event. Preferably the masschanging event is a specific binding event.

The binding event may be a bond making or bond breaking event. Thebinding event may be an associative event (eg formation of a molecularcomposition) or a dissociative event (eg decomposition of a molecularcomposition). The change in molecular density will be specific to thenature of the binding event and the invention can therefore be used toadvantageously differentiate formation of a chemical bond or molecularcomposition of interest from formation of similar chemical bonds ormolecular compositions. In this way, the invention provides a uniquesignature of the molecule or composition which has been formed by thebinding event.

This embodiment is particularly advantageous when the material ofinterest is a complex molecule such as a complex biological molecule (ega protein or DNA). The material of interest may be an antigen and thespecific binding event may be formation of an antibody/antigen specificbinding pair. By way of example, where an antibody is the material ofinterest bound to the sensor component (eg within either a sensingwaveguide or a sensing layer), specific binding of an antigen gives riseto a significant change in molecular density. On the other hand,non-specific binding gives rise to a less significant change inmolecular density. By allowing these effects to be distinguished, thisembodiment of the present invention permits effective integration intobiochemical systems.

In a preferred embodiment of the method of the invention, the masschanging event is a conformational change of the material of interest(eg chemical or biological material of interest). For example, theconformational change may be a molecular rearrangement (eg following abinding event or a change in the ambient environment).

This embodiment advantageously permits materials of interest (egmolecules) which are very similar in nature but have differentconformations to be differentiated eg proteins such as prions or normaland mutant forms of α-synucleins. The material of interest may be aprotein which undergoes a conformational change by a change intemperature, pH or by interaction with an additional species. By way ofspecific example (but not wishing to be bound by theory), hexakinase onthe sensor component (eg within either a sensing waveguide or a sensinglayer) undergoes a conformational change in the presence of glucosegiving rise to a change in molecular density. The present invention issensitive to the molecular density change so as to permit the change inconformation to be determined.

In a preferred embodiment of the method of the invention, the materialof interest is a nucleotide and the mass changing event is nucleotidecomplementation, preferably effective nucleotide complementation (eg theinteraction of DNA sequences in a complementation process or thedetermination of DNA amplification end points). By way of example (butnot wishing to be bound by theory), the material of interest is anucleotide strand on the sensor component (eg within either a sensingwaveguide or a sensing layer) which gives rise to a change in moleculardensity when it undergoes effective nucleotide complementation. Thepresent invention is sensitive to changes in density at a molecularlevel so as to permit effective nucleotide complementation to bedetermined. Thus the degree of effective nucleotide complementation maybe determined.

The mass changing event may be a physical (ie non-chemical) masschanging event. The physical mass changing event may be aggregation.

In a preferred embodiment, step (C) comprises: imposing a condition suchas to induce the mass changing event. The condition may be a chosentemperature, pressure, acidity, solvent or humidity.

Before or after step (B), the method may comprise:

-   -   (1) irradiating the sensor component with electromagnetic        radiation to generate a first output;    -   (2) measuring a characteristic of the first output; and wherein        steps (E) and (F) are:    -   (E) measuring the response of a characteristic of the output        over the temporal range relative to the characteristic of the        first output; and    -   (F) relating the response of the characteristic of the output        over the temporal range relative to the characteristic of the        first output to the mass changing event.

Steps (1) and (2) may be performed at start-up. The results may bestored electronically (eg as calibration data).

In a preferred embodiment, the sensor device is an interferometricsensor device. The sensor component of the interferometric sensor devicemay comprise at least one waveguide (eg a slab or channel waveguide) ora fibre optic component. For example, the sensor component may be awaveguide structure. The waveguide structure may be generally of theplanar type disclosed in WO-A-98/22807 or WO-A-01/36945.

Preferably the sensor component is a waveguide structure including:

-   either (a) one or more sensing layers capable of inducing in a    secondary waveguide a measurable response to a change in the    localised environment caused by the mass changing event or (b) a    sensing waveguide capable of exhibiting a measurable response to a    change in the localised environment caused by the mass changing    event.

In this embodiment, the mass changing event contributes to a change inthe effective refractive index of the sensor component. The waveguidestructure is particularly sensitive to changes in molecular density andthis is advantageously exploited to differentiate mass changing events.

Particularly preferably the sensor component is a waveguide structureincluding:

-   either (a) one or more sensing layers capable of inducing in a    secondary waveguide a measurable response to a change in the    localised environment caused by the mass changing event and an    inactive (eg deactivated) secondary waveguide in which the sensing    layer is incapable of inducing a measurable response to a change in    the localised environment caused by the mass changing event or (b) a    sensing waveguide capable of exhibiting a measurable response to a    change in the localised environment caused by the mass changing    event and an inactive (eg deactivated) waveguide substantially    incapable of exhibiting a measurable response to a change in the    localised environment caused by the mass changing event.

Preferably each of the sensing waveguide or secondary waveguide (or anyadditional waveguides such as reference waveguides) of the sensorcomponent is a planar waveguide (ie a waveguide which permits lightpropagation in any arbitrary direction within the plane). Particularlypreferably each planar waveguide is a slab waveguide.

Preferably the sensor component constitutes a multi-layered structure(eg a laminated waveguide structure) of the types disclosed inWO-A-98/22807 and WO-A-01/36945 (Farfield Sensors Limited). In apreferred embodiment, each of the plurality of layers in themulti-layered sensor component are built onto a substrate (eg ofsilicon) through known processes such as PECVD, LPCVD, etc. Intermediatetransparent layers may be added (eg silicon dioxide) if desired.Typically the sensor component is a multilayered structure of thicknessin the range 0.2-10 microns. A layered structure advantageously permitslayers to be in close proximity (eg a sensing waveguide and an inactive(reference) waveguide may be in close proximity to one another so as tominimise the deleterious effects of temperature and other environmentalfactors). Preferably the sensor component comprises a stack oftransparent dielectric layers wherein layers are placed in closeproximity. Preferably each layer is fabricated to allow equal amounts ofelectromagnetic radiation to propagate by simultaneous excitation of theguided modes in the structure.

The characteristic of the output may be a positional characteristic.Preferably the output is a pattern of interference fringes which may bemeasured (step (E)) by a conventional measuring means (see for exampleWO-A-98/22807) eg one or more detectors such as photodetectors whichmeasure the intensity of electromagnetic radiation. Preferably step (E)comprises: measuring movements in the pattern of interference fringesover the temporal range. Particularly preferably step (E) furthercomprises: calculating the phase shift from the movements in the patternof interference fringes over the temporal range.

The characteristic of the output may be a non-positional characteristic.In a preferred embodiment, the non-positional characteristic of thepattern of interference fringes is the contrast (eg the difference inintensity between the outer fringe envelope and the inner fringeenvelope). For example, the contrast may be the difference in intensitybetween the outer fringe envelope and the inner fringe envelope at acorresponding position in the pattern. Preferably the contrast may bethe difference in intensity between the maxima of the outer fringeenvelope and the maxima of the inner fringe envelope.

The measurement of non-positional characteristics of an interferencepattern is discussed in co-pending UK patent application number0207167.8 of Farfield Sensors Limited.

Preferably the sensor component is adapted so as to be usable inevanescent mode or whole waveguide mode.

Thus in a first embodiment, the sensor component includes one or moresensing layers capable of inducing in a secondary waveguide a measurableresponse to a change in the localised environment caused by the masschanging event. In this first embodiment, the sensor device isadvantageously adapted to optimise the evanescent component so as toinduce in the secondary waveguide a measurable response. The sensorcomponent may comprise a plurality of separate sensing layers to enablemass changing events at different localised environments to bedetermined.

In a preferred embodiment, the sensing layer comprises an absorbentmaterial (eg a polymeric material such as polymethylmethacrylate,polysiloxane, poly-4-vinylpyridine) or a bioactive material (egcontaining antibodies, enzymes, DNA fragments, functional proteins orwhole cells). The absorbent material may be capable of absorbing a gas,a liquid or a vapour containing a chemical material of interest. Thebioactive material may be appropriate for liquid or gas phasebiosensing. For example, the sensing layer may comprise a porous siliconmaterial optionally biofunctionalised with antibodies, enzymes, DNAfragments, functional proteins or whole cells.

In a preferred method of the invention, the secondary waveguidecomprises silicon oxynitride or silicon nitride.

In a second embodiment, the sensor component includes a sensingwaveguide capable of exhibiting a measurable response to a change in thelocalised environment caused by the mass changing event. In this secondembodiment, the sensor device is adapted to minimise the evanescentcomponent and may be used advantageously in whole waveguide mode.

In a preferred embodiment, the sensing waveguide comprises an absorbentmaterial (eg a polymeric material such as polymethylmethacrylate,polysiloxane, poly-4-vinylpyridine) or a bioactive material (egcontaining antibodies, enzymes, DNA fragments, functional proteins orwhole cells). The absorbent material may be capable of absorbing a gas,a liquid or a vapour containing a chemical material of interest. Thebioactive material may be appropriate for liquid or gas phasebiosensing. For example, the sensing waveguide may comprise a poroussilicon material optionally biofunctionalised with antibodies, enzymes,DNA fragments, functional proteins or whole cells.

Where the sensor component comprises a sensing waveguide adapted for usein whole waveguide mode, an absorbent layer in the form of anovercoating may be present for use as a membrane (for example) toseparate out certain stimuli.

To optimise the performance of the first embodiment, the sensorcomponent may further comprise an inactive secondary waveguide in whichthe sensing layer is incapable of inducing a measurable response to achange in the localised environment caused by the mass changing event.The inactive secondary waveguide is capable of acting as a referencelayer. It is preferred that the secondary waveguide and inactivesecondary waveguide have identical properties with the exception of theresponse to the change in the localised environment caused by the masschanging event. By way of example, the secondary waveguide and inactivesecondary waveguide are made of silicon oxynitride.

To optimise the performance of the second embodiment, the sensorcomponent may further comprise an inactive (eg deactivated) waveguidesubstantially incapable of exhibiting a measurable response to a changein the localised environment caused by the mass changing event. Theinactive waveguide is capable of acting as a reference layer. Thephysical, biological and chemical properties of the sensing waveguideand inactive waveguide are as similar as possible (with the exception ofthe response to the change in the localised environment caused by themass changing event). Typically the inactive waveguide is made ofsilicon oxynitride.

As a consequence of mass changing events, changes in the dielectricproperties (eg the effective refractive index) of the sensing waveguideor sensing layer occur. This causes a measurable response (ie a changein the transmission of electromagnetic radiation down the sensingwaveguide (or waveguides) in whole waveguide mode or the secondarywaveguide in evanescent field mode) which (in one embodiment) manifestsitself as a movement of interference fringes. This differs according towhether the sensor component is interrogated in TE or TM mode.

By way of example, the movement of the pattern of interference fringesmay be used to calculate the phase shift which takes place in thesensing waveguide or sensing layer during the passage of electromagneticradiation through the sensor component. The phase shift is effectivelydirectly proportional to changes occurring in the effective refractiveindex of the sensing waveguide or sensing layer and differs according towhether the sensor component is interrogated in TE or TM mode.

A pattern of interference fringes (eg for TE and TM modes respectively)may be generated when the electromagnetic radiation from the sensorcomponent is coupled into free space and may be recorded in aconventional manner (see for example WO-A-98/22807). A response of thesensor component to a change in the localised environment may bemeasured from movement of the fringes in the interference pattern. Thephase shift of the electromagnetic radiation in the sensor component (eginduced in the secondary waveguide in evanescent field mode or exhibitedin the sensing waveguide in whole waveguide mode) may be calculated. Inturn, the change in molecular density contributing to a change in theeffective refractive index may be calculated.

Movement in the interference fringes may be measured either using asingle detector which measures changes in the intensity ofelectromagnetic radiation or a plurality of such detectors which monitorthe change occurring in a number of fringes or the entire interferencepattern. The one or more detectors may comprise one or morephotodetectors (eg photodiodes). Where more than one photodetector isused this may be arranged in an array. In an array format, the relatingmeans capable of relating the measurable response in TM mode and themeasurable response in TE mode to a mass changing event may be deployedin a spatially resolved manner. Such spatial resolution can be achievedby means of (for example) remote imaging or lithography or scanning ameasurement probe as in the case of an atomic microprobe.

In a preferred embodiment of the method of the invention, step (D) iscarried out with electromagnetic radiation in TM mode.

In a preferred embodiment of the method of the invention, step (D) iscarried out with electromagnetic radiation in TE mode.

In a preferred embodiment of the method of the invention, step (D)comprises:

-   (D1) irradiating the sensor component with electromagnetic radiation    in TE mode to produce a first pattern of interference fringes;-   (D2) irradiating the sensor component with electromagnetic radiation    in TM mode to produce a second pattern of interference fringes; and    step (E) comprises:-   (E1) measuring movements in the first pattern of interference    fringes; and-   (E2) measuring movements in the second pattern of interference    fringes.

Particularly preferably step (E) of the method of the invention furthercomprises:

-   (E3) calculating the phase shift of the sensor component in TM mode    from the movements in the first pattern of interference fringes;-   (E4) calculating the phase shift of the sensor component in TE mode    from the movements in the second pattern of interference fringes;-   and step (F) is-   relating the phase shift of the sensor component in TM mode and the    phase shift of the sensor component in TE mode to the mass changing    event.

More preferably step (E) of the method of the invention furthercomprises:

-   (E3) calculating the phase shift of the sensor component in TM mode    from the movements in the first pattern of interference fringes-   (E4) calculating the phase shift of the sensor component in TE mode    from the movements in the second pattern of interference fringes;-   (E5) calculating the phase shift of the sensor component in TM mode    relative to the phase shift of the sensor component in TE mode;-   and step (F) is-   relating the the phase shift of the sensor component in TM mode    relative to the phase shift of the sensor component in TE mode to    the mass changing event.

Preferably the phase shift of the sensor component in TM mode relativeto the phase shift of the sensor component in TE mode is a ratio of thephase shift of the sensor component in TM mode to the phase shift of thesensor component in TE mode.

Step (D) may comprise: generating an output from the sensor component onat least two occasions over a temporal range. Preferably step (D)comprises: generating an output from the sensor component continuouslyover a temporal range.

The mass changing event is typically determined qualitatively but may bedetermined quantitatively in terms of a change in molecular density ormass.

Thus in a preferred embodiment, the method further comprises:

-   (G1) relating the response of the characteristic of the output over    the temporal range to a change in the intrinsic refractive index    and/or the volume; and-   (G2) calculating the change in the molecular density; and-   (G3) optionally calculating the change in mass.

Preferably the method further comprises:

-   (G1) relating the movements in the first pattern of interference    fringes and second pattern of interference fringes to a change in    the intrinsic refractive index and/or the volume; and-   (G2) calculating the change in the molecular density; and-   (G3) optionally calculating the change in mass.

In a particularly preferred embodiment, step (G1) comprises:

(G1) relating the movements in the first pattern of interference fringesand second pattern of interference fringes to a change in the intrinsicrefractive index and/or the thickness of the sensing layer or sensingwaveguide.

Preferably the sensor device further comprises:

-   -   relating means capable of relating the measurable response to a        change in molecular density in the localised environment.

The relating means enables the measurable response to be directlyrelated to the molecular density or indirectly related to the moleculardensity (eg to the dimensionality such as the volume and to the mass)and may be for example a piezoelectric sensing system (where themeasured resonant frequency is related to the mass and the decay timeconstant of free oscillation to the dimension) or an optical system suchas a surface plasmon resonance device in combination with a device basedon other analytical techniques such as ellipsometry.

Where volume is to be measured directly or indirectly, this may be bydetermination of one or more of the relevant dimensions or bydisplacement of the medium surrounding the volume.

In a preferred embodiment of the method, said sensor device furthercomprises:

-   first irradiating means for irradiating the sensor component with    electromagnetic radiation in TM mode;-   second irradiating means for irradiating the sensor component with    electromagnetic radiation in TE mode;-   measuring means for measuring the measurable response of the sensor    component in TM mode and for measuring the measurable response of    the sensor component in TE mode; and-   relating means capable of relating the measurable response of the    sensor component in TM mode and the measurable response of the    sensor component in TE mode to a change in molecular density in the    localised environment.

The first and second irradiating means may be the same or different. Themeasuring means may be one or more detectors in an array.

Preferably the sensor device further comprises:

-   a synchronising means for synchronising the measuring means with the    first irradiating and second irradiating means so as to correlate    the measurement of the measurable response of the sensor component    in TE mode and of the measurable response of the sensor component in    TM mode with the irradiation of the sensor component with    electromagnetic radiation in TE and TM mode respectively.

Particularly preferably the synchronising means is capable ofcalculating the phase shift of the sensor component in TE mode and thephase shift of the sensor component in TM mode.

Particularly preferably the synchronising means is capable of relatingthe movements in the first pattern of interference fringes and secondpattern of interference fringes to the mass changing event.

The first and second irradiating means may be adapted to irradiate thesensor component with electromagnetic radiation in TE or TM modesequentially or simultaneously. The first and second irradiating meansmay be the same or different. Where different sources of electromagneticradiation are used, an optical switch (eg a rotating mirror) may be usedto switch rapidly between the two. Alternatively, a single source ofelectromagnetic radiation may be used to simultaneously excite TE and TMmodes of the sensor component by (for example) aligning the polarisationvector of the linearly polarised source at an angle with respect to theplane of the sensing waveguide or sensing layer of the sensor component.An active analyser system may be used to alternately remove the unwantedTE or TM mode radiation during data capture of the desired TM or TEoutput respectively. The active analyser system may comprise anelectro-optic half wave plate placed in series with an analyser.

In a preferred embodiment, an adjustable analyser may be used to measurethe first pattern of interference fringes and the second pattern ofinterference fringes separately. The measurements may be synchronisedwith the excitation and/or polarisation procedure to ensure that phaseshift information is correlated with TE and TM excitation.

A controller may be provided to synchronise the one or more sources ofelectromagnetic radiation and one or more detectors. For example, thecontroller may capture the data from a photodetector (eg photodiode)array. The firing of the (or each) source of electromagnetic radiationmay be synchronised by the controller with the alternate capture of thefirst and second pattern of interference fringes generated in TM modeand TE mode. The controller may be adapted to calculate the phase shiftin TE and TM modes independently.

Electromagnetic radiation generated from a conventional source may bepropagated into the sensor component in a number of ways. In a preferredembodiment, electromagnetic radiation is simply input via an end face ofthe sensor component (this is sometimes described as “an end firingprocedure”). Preferably the electromagnetic radiation source providesincident electromagnetic radiation having a wavelength falling withinthe optical range. Propagating means may be employed for substantiallysimultaneously propagating incident electromagnetic radiation into aplurality of waveguides. For example, one or more coupling gratings ormirrors may be used. A tapered end coupler rather than a couplinggrating or mirror may be used to propagate radiation into the lowermostwaveguide. Preferably the amount of electromagnetic radiation in thesensing waveguide/inactive waveguide or in the secondarywaveguide/inactive secondary waveguide is equal.

The incident electromagnetic radiation may be oriented (eg planepolarised) as desired using an appropriate polarising means. Theincident electromagnetic radiation may be focussed if desired using alens or similar micro-focussing means.

A plurality of electromagnetic radiation detector units (eg in an array)and/or a plurality of electromagnetic radiation sources may be used tomeasure in discrete areas of the sensor component simultaneously theresponses to changes in the localised environment caused by the masschanging event. Alternatively, the position of the electromagneticradiation detector and electromagnetic radiation source relative to thesensor component may be changed to provide information concerningresponses in discrete areas of the sensor component. For example,discrete responses to a change in the localised environment caused bydifferent mass changing events may be measured in discrete areas of thesensor component. For this purpose, the preferred assembly makes use ofthe versatility of the evanescent mode and comprises a plurality ofseparate sensing layers or regions.

The sensor component may be excited across its width and atwo-dimensional photodiode array (or the like) may be used toeffectively interrogate “strips” of the sensor component (eg an arraysensor). This may be carried out across more than one axissimultaneously or sequentially to provide spatially resolved informationrelating to mass changing events on the sensor component.

Preferably the sensor device comprises:

-   -   means for intimately exposing at least a part of the (or each)        sensing layer or the sensing waveguide of the sensor component        to the localised environment.

In a preferred embodiment, the means for intimately exposing at least apart of the sensing layer or the sensing waveguide to the localisedenvironment is integrated onto the sensor component.

Preferably the means for intimately exposing at least a part of the (oreach) sensing layer or the sensing waveguide of the sensor component tothe localised environment is as described in WO-A-01/36945. The meansmay be automated in order to reduce the requisite degree of userintervention.

Preferably the means for intimately exposing at least a part of thesensing layer or the sensing waveguide to the localised environment isadapted to permit the continuous introduction of an analyte containing amaterial of interest (ie a dynamic system). For example, it may permitthe continuous introduction of the material of interest in adiscontinuous flow (eg as a train of discrete portions) into thelocalised environment. This may be achieved by capillary action or by aseparate urging means.

Preferably the means for intimately exposing at least a part of thesensing layer or the sensing waveguide to the localised environment isadapted to induce mass changing events (eg reactions) in a staticanalyte containing a chemical or biological material of interest. Inthis sense, the system may be considered to be dynamic. Mass changingevents (eg reactions) may be induced in any conventional manner such asby heat or radiation.

The means for intimately exposing at least a part of the (or each)sensing layer or the sensing waveguide to the localised environment maybe a part of a microstructure positionable on the surface of and inintimate contact with the sensor component.

Preferably the microstructure comprises means for intimately exposing atleast a part of the sensing layer or the sensing waveguide to thelocalised environment in the form of one or more microchannels and/ormicrochambers. For example, an analyte containing a chemical material ofinterest may be fed through microchannels or mass changing events (egchemical reactions) may take place in an analyte containing a materialof interest located in a microchamber. An analyte containing chemicalmaterial of interest may be fed into the microchannels by capillaryaction or positively fed by an urging means.

In a preferred embodiment, the means for intimately exposing at least apart of the (or each) sensing layer or the sensing waveguide to thelocalised environment is included in a cladding layer. For example,microchannels and/or microchambers may be etched into the claddinglayer. The cladding layer may perform optical functions such aspreventing significant discontinuities at the boundary of the sensingwaveguide or sensing layer(s) or chemical functions such as restrictingaccess of certain species to the sensing waveguide or sensing layer(s).The cladding layer may be integrated onto the sensor component.

Preferably the whole of or a portion of any additional functionality maybe included in the cladding layer. In one embodiment, the sensing layeritself may be incorporated in the cladding layer (for example in theform of an absorbent material). Particularly preferably, the whole ofthe additional functionality may be provided in the cladding layer andinclude sensing devices such as for example quadrature electric fieldtracks or other microfluidic sensing devices. The cladding layer mayincorporate an electromagnetic source (eg a laser) and/or means fordetecting electromagnetic radiation (of the type detailed below). Thecladding layer may incorporate a chemical separating means (eg an HPLCbased device).

Preferably the means for exposing at least a part of the (or each)sensing layer or the sensing waveguide of the sensor component to thelocalised environment is a sensor cell. Preferably the sensor cell has alow volume (eg 50 microlitres or less). By keeping the cell volumesmall, the method of the invention is not hampered and the amount ofprotein consumed is advantageously minimised.

The present invention will now be described in a non-limitative sensewith reference to the Examples and accompanying Figures in which:

FIG. 1 represents schematically in plan view a sensor device for use inan embodiment of the invention;

FIG. 2 represents schematically in plan view a sensor device for use inan embodiment of the invention;

FIG. 3 represents schematically in plan view a sensor device for use inan embodiment of the invention;

FIG. 4 illustrates 20 oligonucleotide DNA hybridisation using a AnaLightBio250 (Farfield Sensors Limited);

FIG. 5 illustrates a study of a metabolic pathway on the AnaLightBio250;

FIG. 6 illustrates schematically the sensor component used in theExamples; and

FIG. 7 illustrates TE vs TM for determining a unique combination ofintrinsic refractive index and thickness.

A sensor device for performing an embodiment of the method of theinvention is shown schematically in FIG. 1. Plane polarisedelectromagnetic radiation is generated in TM mode using a firstelectromagnetic radiation source (11) and in TE mode using a secondelectromagnetic radiation source (12) relative to the sensor component(13). The sensor component (13) is of a multi-layered type as describedin WO-A-98/22807 and illustrated in FIG. 6.

An interference pattern is captured by the photodiode or photodiodearray (14). The two electromagnetic radiation sources (11 and 12) arecontrolled electronically by a controller (15) that also captures thedata from the photodiode array (14). The firing of each of theelectromagnetic radiation sources (11) and (12) is synchronised by thecontroller (15) such that the interference patterns in TM mode and TEmode are alternately captured. The controller (15) calculates the phaseshift in each mode and the TE and TM phase shifts are compared in orderto measure the change in molecular density. This data may be reporteddirectly or made available to secondary systems for further analysis.

A further embodiment is shown schematically in FIG. 2 in which arotating mirror (26) is employed to switch rapidly betweenelectromagnetic radiation in TE and TM mode from first and secondelectromagnetic radiation sources (21) and (22) respectively forirradiating sensor component (13). A controller (25) is arranged tosynchronise the electromagnetic radiation sources (21) and (22), theoptical switch (26) and the photodiode array (24).

In FIG. 3, a single electromagnetic radiation source (31) is used tosimultaneously irradiate the sensor component (13) with electromagneticradiation in TE and TM modes. This is achieved by aligning thepolarisation vector of the linearly polarised optical source at an anglewith respect to the plane of the sensing layer or sensing waveguide ofthe sensor component (13). An active analyser system (37) is used toremove unwanted output of TE or TM radiation during capture of theoutput of desired TM or TE radiation respectively. This may consist ofan electro-optic half wave plate placed in series with an analyser. Acontroller (35) is arranged to synchronise the source (31), the analysersystem (37) and the photodiode array (34).

The interferometric sensor component (13) of the sensor device used inthe following Examples is illustrated in FIG. 6. The interferometricsensor component (13) is a laminate structure consisting of an absorbentsensing layer (1) separated from a reference waveguide (3) by a silicondioxide spacer (2). A further silicon dioxide spacer (4) separates thesilicon oxynitride reference waveguide (3) from a substrate (5) ofsilicon.

EXAMPLE 1 Immobilisation of Pre-Silanised Oligonucleotides

[(3-mercaptopropyl)-trimethoxysilane] was diluted to 5 mM stock solutionin sodium acetate buffer (30 mM, pH 4.3). 3 μl of thiol-labelled oligo(thiol, 3 nMol) were incubated with 3 μl 5 mM mercaptosilane solutionand 54 μl sodium acetate buffer for 2 hours at room temperature. Sensorcomponents were immersed in 10% NaOH for 30 minutes, washed thoroughlywith distilled water and dried for at least 15 minutes at 80° C.

In order to chemically attach the oligonucleotide strand to the sensorcomponent surface, the silanised nucleic acids were pipetted on thesensor components, incubated for 15 minutes at room temperature anddried at 50° C. for 5 minutes. The sensor components were dipped in hotwater (80° C., 30 s) to remove non-covalently bound oligonucleotides anddried at 50° C.

To investigate the unspecific binding of non-complementaryoligonucleotide, 200 μl of 1 μM oligo 7A3H were injected into the sampleloop. After complete coverage of the sensor component surface with testsolution the flow was stopped for 20 minutes.

Prior to hybridisation of complementary oligonucleotide, non tightlybound non-complementary oligonucleotide was removed by washing thesurface with 2×SSC until a stable baseline was reached. Subsequently,200 μl complementary strand RP4T was injected and incubated for 20minutes on the sensor surface. This was followed by a washing step withrunning buffer. 50 mM NaOH was applied to denature the double-strand onthe surface and to make it available for a second hybridisationreaction.

The onset of hybridisation is shown in FIG. 4 where the complimentarystrand is added at 2500 s. This binding is stable since it is notreversed when oligonucleotide free buffer solution is washed over thesensor component at 4000 s.

Changing the pH or raising the temperature disrupts the hybridisation.As seen in FIG. 4, sodium hydroxide solution is added at 6300 s andflushed through thereby removing the hybridised complimentary strand andeffectively regenerating the surface.

FIG. 4 shows that the TE response is larger than the TM response. Thisimplies a significant structural change in the surface morphology of thefilm (ie a reduction in thickness) as one would expect from theformation of a hybridised, double helical structure collapsing onto thesurface of the sensor component. Furthermore, the response is of theopposite sense to that of non-specific molecular attachment in which theTM response would be larger than the TE response.

The effect of the decrease in thickness with the increase in massassociated with the specific binding event results in a substantialincrease in the molecular density of the sensing layer. Non-specificmolecular attachment by comparison results in an increase in both layerthickness and mass and result in substantially the same moleculardensity. Sensitivity of the sensing layer to the molecular density (asopposed to the mass) allows differentiation between specific andnon-specific binding and greatly increases the signal to noise ratio andtherefore the utility of the sensor device (probe). This is generallytrue for any binding event but is especially so in the case of DNAhybridisation where the creation of the double helix structure is one ofthe largest changes in molecular density observable. A more generalexample of specific and non-specific binding is described below.

EXAMPLE 2 Determination of Specific and Non-Specific Binding in aMetabolic Pathway

Fatty acid biosynthesis in plants is carried out by a number of solubleenzymes with each protein molecule containing a single activity.However, there is increasing evidence that consecutive enzymes in themetabolic sequence interact in vivo so that chemical intermediates arepassed between them in a process known as substrate channelling.

A detailed understanding of the mechanisms involved in fatty acidsynthesis will enable these processes to be manipulated. Plants arealready used as bioreactors for the synthesis of useful molecules andfoodstuffs which are harvested from the plants. By either disrupting thesynthetic pathway (or perhaps by substituting an alternative protein)the range and quantities of these plant products could be expanded toinclude useful compounds that are at present not made in specific plantsor to improve the yield of those that are.

FIG. 5 shows the immobilization of enolreductase (ENR—an enzyme involvedin fatty acid biosynthesis) on a chemically activated sensor componentat 400 s. The surface of the sensor component is then blocked withlysozyme at 1700 s. At 2600 s, Cro-acp (the enzyme's substrate) is firstintroduced. This is not expected to bind without the co-factor NAD+ butseems to do so, demonstrating a non-specific event. At 3600 s, NAD+ isadded and some of this small molecule binds to the structure. WhenCro-acp is added again, more binds to the surface.

With the benefit of TE and TM mode electromagnetic irradiation, thesequence of events occurring during this experiment can be qualitativelyinterpreted. ENR forms as a film on the surface, the lysozyme can beseen to sorb into the film and is mostly washed away again. The firstCro-acp does not change the molecular density of the film very much (theTE/TM ratio is constant) but addition of the second aliquot (afteraddition of the NAD+) causes a noticeable densification of the film (theTE/TM ratio reduces sharply). Non specific adhesion can therefore bedistinguished from the specific binding generated by the introduction ofthe co-factor NAD+.

EXAMPLE 3 Analysis of Slab Waveguide Structures Using EM Field Theory

Generally, the intrinsic refractive index and thickness can be resolvedfrom the measurement of effective refractive index by the solution ofMaxwell's equations for propagation of electromagnetic radiation throughthe combined sensing system and analyte.

A—Three Layer Waveguide Structure

For a general three layer (slab) waveguide structure, it is possible toderive an eigenvalue equation for each polarisation. For TE modes:α₁ t=tan⁻¹(α₂/α₁)+tan⁻¹(α₃/α₁)+mπ (m=0, 1, 2 . . . )where m is the mode order andα₁ ²=(n ₁ ² k ₀ ²−β_(m) ²); α₃ ²=(β_(m) ² −n ₃ ² k ₀ ²); α₂ ²=(β_(m) ²−n ₂ ² k ₀ ²) and β_(m) =n _(eff,m) k ₀.(where the core layer is 1, substrate and cladding layers 2 and 3,thickness of the core layer t and the free space wavenumber$\left. {k_{0} = \frac{2\pi}{\lambda_{0}}} \right).$

For TM mode the equation is:${\alpha_{1}t} = {{\tan^{- 1}\left( {\frac{n_{1}^{2}}{n_{2}^{2}}{\alpha_{2}/\alpha_{1}}} \right)} + {\tan^{- 1}\left( {\frac{n_{1}^{2}}{n_{3}^{2}}{\alpha_{3}/\alpha_{1}}} \right)} + {m\quad\pi}}$

Generally, these equations are solved for the effective refractive indexvalues by numerical techniques given a known set of refractive index andthickness parameters. Alternatively if the absolute effective indexvalue is known, it is possible to solve for one variable of the systemfor each equation given that the other three are known.

In the sensor, the starting effective refractive index value(n_(eff)(0)) is known since the intrinsic refractive index and thicknessparameters of the system are known. This would be obtained by numericalsolving of either of the above for the relevant polarisation. The sensorwould then record an effective refractive index difference (Δn_(eff))upon a new cladding layer being introduced (eg a binding event). Anabsolute effective index n_(eff)(1)=n_(eff)(0)+Δn_(eff) could be put into either of the two equations above to solve for the refractive indexof the cladding layer.

B—Four and More Layer Waveguide Structure

To analyse more complicated structures where the number of parameterscan be arbitrarily large requires matrix methods to reduce thecomplexity. The equation to solve becomes:j(α_(s) m ₁₁+α_(c) m ₂₂)=m ₂₁−α_(s)α_(c) m ₁₂where α_(s) and α_(c) refer to the terms for substrate and cladding, thetwo outer bounding layers, within which the field has a decayingexponential form. They are written as:α_(s) ²=(β_(m) ² −n _(s) ² k ₀ ²) and α_(c) ²=(β_(m) ² −n _(c) ² k ₀ ²)

The m_(ij) terms are elements of a 2×2 matrix which is the resultantmatrix formed by multiplication of the individual characteristic matrixof each layer. Thus for each layer within the stack (not the boundinglayers) we have for TE mode; $M_{i} = {{\begin{matrix}{\cos\quad\left( {\alpha_{i}t_{i}} \right)} \\{j\quad\alpha_{i}{\sin\left( {\alpha_{i}t_{i}} \right)}}\end{matrix}\begin{matrix}{{j/\alpha_{i}}\sin\quad\left( {a_{i}t_{i}} \right)} \\{\cos\quad\left( {\alpha_{i}t_{i}} \right)}\end{matrix}}}$where t_(i) is the thickness of each layer, and the index of each layeris;α_(i) ²=(n _(i) ² k ₀ ²−β_(m) ²)

The characteristic matrix (M) for the whole stack is obtained throughmultiplication as follows; $M = {{{\begin{matrix}m_{11} \\m_{21}\end{matrix}\begin{matrix}m_{12} \\m_{22}\end{matrix}}} = {{M_{1} \cdot M_{2} \cdot M_{3} \cdot \ldots}\quad M_{n}}}$For TM modes we modify the terms in the characteristic matrix for eachlayer

The equation must be initially solved numerically using a knownstructure and calculating the TE effective index. The sensor would thenprovide a new TE effective index after layer deposition. This would befed into the multilayer equation and solved for one parameter (eitherthickness or refractive index) of the film, setting the other parameteras a range variable. The analogous process could be carried out usingthe corresponding TM result. The unique combination of intrinsicrefractive index and thickness would be found by correlating the TE andTM results as shown in FIG. 7. The crossing point is the unique solutionfor intrinsic refractive index and thickness for the combined sensor andanalyte system.

From the intrinsic refractive index one can determine density and theabsolute thickness is directly related to volume from which the absolutemass follows (density×volume=mass). Furthermore it is generally knownthat proteins may be discriminated by mass which can be determined fromthe calculated density if the thickness is determined as per theprevious example.

Although intrinsic refractive index is not directly related to density,if we make certain assumptions regarding the system under investigation(eg water has zero protein density at a refractive index of 1.33 and aknown protein (eg streptavidin) has a refractive index of 1.45 for aknown density) then generally one can attribute a change in refractiveindex to a change in percentage protein density:${{Density}\quad\left( {{unknown}\quad{protein}} \right)} = \frac{\begin{matrix}{{Density}\quad\left( {{protein}\quad{standard}} \right)*} \\\left( {{{RI}\quad\left( {{unknown}\quad{protein}} \right)} -} \right. \\\left. {{RI}\quad({water})} \right)\end{matrix}}{\begin{matrix}\left( {{{RI}\quad\left( {{protein}\quad{standard}} \right)} -} \right. \\\left. {{RI}\quad({water})} \right)\end{matrix}}$Absolute molecular density is a fundamental characteristic of anymolecule and its determination and discrimination can therefore be usedas a differentiating molecular signature.

1. A method for determining a mass changing event in a material ofinterest in a localised environment, said method comprising: (A)providing a sensor device having a sensor component capable ofexhibiting a measurable response to a change in the localisedenvironment caused by the mass changing event in the material ofinterest therein; (B) introducing the material of interest into thelocalised environment; (C) inducing the mass changing event in thematerial of interest; (D) generating an output from the sensor componentover a temporal range; (E) measuring the response of a characteristic Ofthe output over the temporal range; and (F) relating the response of thecharacteristic of the output over the temporal range to the masschanging event.
 2. A method as claimed in claim 1 wherein step (D)comprises: irradiating the sensor component with electromagneticradiation to generate an output over a temporal range.
 3. A method asclaimed in claim 1 wherein the mass changing event is a chemical masschanging event.
 4. A method as claimed in claim 3 wherein the masschanging event is a specific molecular (or atomic) interaction.
 5. Amethod as claimed in claim 4 wherein the mass changing event is aspecific associative or dissociative molecular interaction.
 6. A methodas claimed in claim 1 wherein the mass changing event is a bindingevent.
 7. A method as claimed in claim 1 wherein the mass changing eventis a specific binding event.
 8. A method as claimed in claim 1 whereinthe binding event is a bond making or bond breaking event.
 9. A methodas claimed in claim 1 wherein the binding event is an associative eventor a dissociative event.
 10. A method as claimed in claim 9 wherein thebinding event is formation of a molecular composition or decompositionof a molecular composition.
 11. A method as claimed in claim 6 whereinthe material of interest is a biological molecule.
 12. A method asclaimed in claim 11 wherein the biological molecule is an antigen andthe binding event is formation of an antibody/antigen specific bindingpair.
 13. A method as claimed in claim 1 wherein the mass changing eventis a conformational change of the material of interest.
 14. A method asclaimed in claim 13 wherein the conformational change is a molecularrearrangement.
 15. A method as claimed in claim 1 wherein the materialof interest is a nucleotide and the mass changing event is effectivenucleotide complementation.
 16. A method as claimed in claim 1 whereinthe mass changing event is a physical mass changing event.
 17. A methodas claimed in claim 16 wherein the mass changing event is aggregation.18. A method as claimed in claim 1 wherein step (C) comprises: imposinga condition such as to induce the mass changing event.
 19. A method asclaimed in claim 18 wherein the condition is selected from the groupconsisting of a chosen temperature, pressure, acidity, solvent andhumidity.
 20. A method as claimed in claim 1 wherein the sensor deviceis an interferometric sensor device.
 21. A method as claimed in claim 20wherein the sensor component is a waveguide structure including: either(a) one or more sensing layers capable of inducing in a secondarywaveguide a measurable response to a change in the localised environmentcaused by the mass changing event or (b) a sensing waveguide capable ofexhibiting a measurable response to a change in the localisedenvironment caused by the mass changing event.
 22. A method as claimedin claim 20 wherein the sensor component is a waveguide structureincluding: either (a) one or more sensing layers capable of inducing ina secondary waveguide a measurable response to a change in the localisedenvironment caused by the mass changing event and an inactive secondarywaveguide in which the sensing layer is incapable of inducing ameasurable response to a change in the localised environment caused bythe mass changing event or (b) a sensing waveguide capable of exhibitinga measurable response to a change in the localised environment caused bythe mass changing event and an inactive waveguide substantiallyincapable of exhibiting a measurable response to a change in thelocalised environment caused by the mass changing event.
 23. A method asclaimed in claim 20 wherein each of the sensing waveguide or secondarywaveguide of the sensor component is a planar waveguide.
 24. A method asclaimed in claim 1 wherein the mass changing event contributes to achange in the effective refractive index of the sensor component.
 25. Amethod as claimed in claim 20 wherein the characteristic of the outputis a positional characteristic.
 26. A method as claimed in claim 25wherein the output is a pattern of interference fringes.
 27. A method asclaimed in claim 26 wherein step (E) comprises: measuring movements inthe pattern of interference fringes over the temporal range.
 28. Amethod as claimed in claim 27 wherein step (E) further comprises:calculating the phase shift from the movements in the pattern ofinterference fringes over the temporal range.
 29. A method as claimed inclaim 20 wherein the characteristic of the output is a non-positionalcharacteristic.
 30. A method as claimed in claim 29 wherein thenon-positional characteristic of the pattern of interference fringes isthe contrast.
 31. A method as claimed in claim 2 wherein step (D) iscarried out with electromagnetic radiation in TM mode.
 32. A method asclaimed in claim 2 wherein step (D) is carried out with electromagneticradiation in TE mode.
 33. A method as claimed in claim 2 wherein step(D) comprises: (D1) irradiating the sensor component withelectromagnetic radiation in TE mode to produce a first pattern ofinterference fringes; (D2) irradiating the sensor component withelectromagnetic radiation in TM mode to produce a second pattern ofinterference fringes; and step (E) comprises: (E1) measuring movementsin the first pattern of interference fringes; and (E2) measuringmovements in the second pattern of interference fringes.
 34. A method asclaimed in claim 33 wherein step (E) further comprises: (E3) calculatingthe phase shift of the sensor component in TM mode from the movements inthe first pattern of interference fringes; (E4) calculating the phaseshift of the sensor component in TE mode from the movements in thesecond pattern of interference fringes; and step (F) is relating thephase shift of the sensor component in TM mode and the phase shift ofthe sensor component in TE mode to the mass changing event.
 35. A methodas claimed in claim 33 wherein step (E) further comprises: (E3)calculating the phase shift of the sensor component in TM mode from themovements in the first pattern of interference fringes (E4) calculatingthe phase shift of the sensor component in TE mode from the movements inthe second pattern of interference fringes; (E5) calculating the phaseshift of the sensor component in TM mode relative to the phase shift ofthe sensor component in TE mode; and step (F) is relating the phaseshift of the sensor component in TM mode relative to the phase shift ofthe sensor component in TE mode to the mass changing event.
 36. A methodas claimed in claim 35 wherein the phase shift of the sensor componentin TM mode relative to the phase shift of the sensor component in TEmode is a ratio of the phase shift of the sensor component in TM mode tothe phase shift of the sensor component in TE mode.
 37. A method asclaimed in claim 33 further comprising: (G1) relating the movements inthe first pattern of interference fringes and second pattern ofinterference fringes to a change in the intrinsic refractive indexand/or the volume; and (G2) calculating the change in the moleculardensity; and (G3) optionally calculating the change in mass.
 38. Amethod as claimed in claim 37 wherein step (G1) comprises: (G1) relatingthe movements in the first pattern of interference fringes and secondpattern of interference fringes to a change in the intrinsic refractiveindex and/or the thickness of the sensing layer or sensing waveguide.39. A method as claimed in claim 20 wherein the sensor device furthercomprises: means for intimately exposing at least a part of the (oreach) sensing layer or the sensing waveguide of the sensor component,said means defining the localised environment having a volume of 50microlitres or less.
 40. A method as claimed in claim 1 wherein step (D)comprises: generating an output from the sensor component on at leasttwo occasions over a temporal range.
 41. A method as claimed in claim 40wherein step (D) comprises: generating an output from the sensorcomponent continuously over a temporal range.
 42. A method as claimed inclaim 1 wherein step (D) comprises: electromechanically vibrating thesensor component to generate an output over a temporal range.
 43. Amethod as claimed in claim 42 wherein the sensor component is a quartzcrystal.
 44. A method as claimed in claim 1 wherein step (D) comprises:irradiating the sensor component with energetic particles to generate anoutput over a temporal range.
 45. A method as claimed in claim 44wherein the energetic particles are neutrons, α-particles orβ-particles.